The identification of airway stem cells is a critical aspect in the development of gene therapies for diseases such as cystic fibrosis (CF). Furthermore, the complexities of disease pathophysiology in CF have suggested that submucosal glands may be important therapeutic targets. One approach to target this region, which is inaccessible from the airway lumen, is to facilitate gene transfer to airway stem cells responsible for submucosal gland development in utero. However, at present little is known about the biology of these stem cell targets in the airway. To this end, we propose to characterize the cellular phenotype of airway stem cells using molecular approaches that evaluate critical aspects of gene regulation important in the formation of submucosal glands. Experiments will attempt to identify and characterize the developmental pathways leading to stem cell commitment in submucosal gland morphogenesis. Such developmental mechanisms likely also play important roles in other hypersecretory diseases such asthma and chronic bronchitis, where submucosal gland hyperplasia and/or hypertrophy occur. Previous studies by this laboratory using retroviral based lineage analysis in the airway have suggested that a pluripotent surface airway epithelial stem cell compartment also has the ability to form submucosal glands. However, relatively little is currently known about the molecular phenotype of these airway stem cells. We will evaluate in detail the regulation of one particular gene, lymphoid enhancing factor-1 (Lef-1), a transcription factor known to regulate cell-fate decisions in other tissues. Recently we showed that activation of Lef-1 gene expression defines an airway progenitor/stem cell compartment at the earliest stages of epithelial commitment to form submucosal glands (ie., epithelial condensation involved in gland bud formation). Using Lef-1 as a molecular marker for this stem cell compartment, we propose to delineate pathways and transcription factors involved in its regulation using ferret and transgenic mouse models of the airway. Detailed analysis of the Lef-1 promoter will be performed using novel in vivo based model systems that include both recombinant gutted adenoviral vectors, as well as more traditional approaches involving transgenic mice. Lastly, this proposal will also attempt to evaluate mechanisms of Lef-1 action in submucosal gland morphogenesis. Of specific interest is the pathway involving wnt activation of beta-catenin, which is known to be a co-factor for Lef-1 in some, but not all, tissues. We will evaluate a possible role for the wnt pathway and beta-catenin in submucosal gland development using a novel airway specific transgenic mouse model expressing Lef-1 mutants incapable of binding with beta-catenin. Ultimately, this project will increase our understanding of stem cell phenotypes in the airway that have pluripotent capacity for submucosal gland development. Such information will undoubtedly be useful in the development of gene therapies targeting airway stem cells and submucosal glands. Additionally, an increased understanding of submucosal gland developmental mechanisms may provide new therapeutic approaches for treating submucosal gland hyperplasia and hypertrophy in hypersecretory diseases.